Treatment of age-related macular degeneration

ABSTRACT

The present invention relates to a method and compositions for the treatment of age-related macular degeneration (AMD), in particular dry-AMD, specifically geographic atrophy (GA) or advanced dry-AMD. Specifically, the invention relates to an inner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tight junction protein and/or a circadian clock protein for use in the prevention and/or treatment of age-related macular degeneration.

FIELD OF THE INVENTION

The present invention relates to a method and compositions for thetreatment of age-related macular degeneration (AMD), in particulardry-AMD, specifically geographic atrophy (GA) or advanced dry-AMD.

BACKGROUND OF THE INVENTION

Globally, an estimated 161 million people are visually handicapped,among which 37 million fall within the category of legal blindness,generally accepted to have visual acuity of <3/60 or correspondingvisual field loss to <10 degrees. The most common causes of visualhandicap are cataract and glaucoma. However, in the developed world,age-related macular degeneration (AMD) is the most prevalent cause ofregistered blindness in the older population, closely followed bydiabetic retinopathy.

Epidemiological studies of AMD have been undertaken in at least ninecountries. In one study from the United States (US), 1.75 million peoplewere estimated to have advanced AMD defined as geographic atrophy (GA)or neovascularisation in at least one eye, with 7.3 million people atrisk of developing the disease owing to the presence of drusen, tinyyellow or white accumulations of extracellular material that build upbetween Bruch's membrane and the retinal pigment epithelium of the eye.In another study from the United Kingdom (UK), it was found that thereare currently 172,000 and 245,000 people with GA and neovascular AMDrespectively, while a study from Germany found that there are 710,000cases of advanced AMD, predicted to increase to over one million by2020. Given that, in the overall, only limited therapies are availablefor these diseases, their negative social and economic impact isimmense. The cost of AMD, involving diagnosis, monitoring, visual aids,habitation, accident treatment, rehabilitation, treatment of associateddepression and anxiety, as well as direct treatment of the diseaseitself has been estimated to amount to approximately €200,000 perpatient in any five year period.

AMD is the leading cause of central retinal vision loss worldwide.Drusen accumulation is the major pathological hallmark common to bothdry and wet AMD. While numerous mechanisms have been proposed and someimplicated in disease progression; to date, the pathways leading to theend stage of the condition remain unclear. It is estimated that 1 in 10people over the age of 55 show early signs of AMD. In 10% of patientswith AMD, blood vessels sprout from underlying choroidal vasculaturedisrupting retinal tissue integrity, leading to vision loss. Althoughless common, the neovascular form of the disease, termed “wet” AMD, isthe most severe form and is termed a priority eye disease by the WorldHealth Organization (WHO).

The major early pathological hallmark common to both dry and wet AMD isthe accumulation of drusen behind the retina between the RPE and thechoroid. Drusen is composed of extracellular components including, butnot limited to, amyloid-β, vitronectin, cholesterols and almost everycomplement component. These components have all previously beenidentified in several tissues including blood and photoreceptors, and itis abundantly obvious that AMD is primarily a condition that involvesaberrant clearance mechanisms. What is less clear however is the sourceof drusen and the dynamic events that occur during and after the diurnalshedding of photoreceptor outer segments and subsequent phagocytosis bythe retinal pigment epithelium (RPE). On a daily basis, RPE cellsphagocytose photoreceptor outer segments (POS) that are shed duringrenewal of photoreceptors. While some of the phagocytosed material isrecycled to replenish essential components of the photoreceptors, othercomponents in the material are exocytosed to the basolateral compartmentof the RPE and are likely cleared by the systemic immune system. This isa highly regulated process controlled by autophagic processes anddysfunctional rates of clearance are likely a significant contributingfactor to drusen accumulation in some individuals as evidenced byresidual body build up in lysosomes observed in the RPE of AMD donoreyes.

While drusen deposition and localisation can differ from individual toindividual, it is pertinent to consider that there is a considerabledegree of symmetry in drusen patterning in each eye of a singleindividual. This correlates with an equally high degree of interocularsymmetry of retinal blood vessels between right and left eyes. Theseblood vessels which form the inner blood-retina barrier (iBRB) arecritical to maintaining retinal homeostasis. Endothelial cells that linethese vessels have evolved tight junctions, a series of up to 30interacting proteins that limit the paracellular space betweenendothelial cells to all but the smallest of molecules. As well asregulating the exchange of ions and macromolecules between the blood andthe delicate neural microenvironment, these highly specializedendothelial cells protect the retina by restricting the entry ofpotentially damaging blood-borne agents such as neurotoxic chemicals,antibodies, pathogens, immune cells and anaphylatoxins. They alsoexpress a variety of transporters to control both the selectivetransport of nutrients into the retina and the efflux of metabolites andtoxins from the retina via the transcellular pathway.

The end stage of “dry” AMD is termed geographic atrophy (GA), where theRPE begins to degenerate in the region of the macula and can eventuallylead to death of cone photoreceptor cells and eventually central retinalvision loss. GA is primarily a disorder of a cell type at the back ofthe eye called the retinal pigment epithelium (RPE) and the presentinvention can regulate the flux of material into and out of the RPE,thereby relieving the stress exerted on this cell and preventingultimate cell death. Given the pervasive nature of AMD in the developedworld and the wealth of research in this area, the underlying molecularpathology associated with GA development is still far from clear. Thereare currently no therapies on the market to prevent or slow the courseof development of this form of blindness other than recommendedlifestyle changes such as smoking cessation and dietary modification.Supplementation in the diet with multi-vitamins enriched with theanti-oxidants lutein and xeaxanthin have been reported to increasemacular pigment which may be beneficial to patients with dry AMD.

In 2006, the FDA approved the use of Genentech's drug Lucentis® for usein the exudative form of AMD where choroidal neovascularisation (CNV) isthe hallmark pathology, While Lucentis® is indicated for use in thetreatment of wet AMD, a highly similar drug, also originallymanufactured by Genentech, Avastin®, is used “off-label” for thetreatment of wet AMD. Recently, Regeneron Pharmaceuticals, Inc. releasedEylea®, which is a fusion protein that acts as a decoy receptor for thevascular endothelial growth factor receptor 2 (VEGFR2). However, thesedrugs are prohibitively expensive with Lucentis® and Eyelea®, having acost of approximately €1,200-€1,500 per injection, while Avastin® costsapproximately €110 per injection. Thus, there remains a need to developalternative therapies for AMD, both wet and dry AMD.

The mammalian circadian clock in the neurons of suprachiasmatic nuclei(SCN) in the brain and in cells of peripheral tissues is driven by aself-sustained molecular oscillator, which generates rhythmic geneexpression with a periodicity of about 24 hours. The 24-hour circadiancycle is controlled by the oscillatory expression of a clock genecassette including Period (Per) 1-3, Cryptochrome (Cry) 1/2, Clock, andBmal1/2.

Circadian clocks influence nearly all aspects of physiology andbehaviour, including rest-wake cycle, cardiovascular activity, hormonesecretion, body temperature, and metabolism. Circadian rhythms areconserved across species even in nocturnal animals. All of the circadianclock components cycle identically in every species from Drosophilathrough mouse to man. While up to 40% of genes can cycle in a circadianfashion, very few genes display this characteristic cycling in alltissue with the same rhythm. Intriguingly, if one clock component is outof synchronisation, the entire system is at risk of failing which canlead to a wide and varied amount of phenotypes in mammalian systems. Forexample, Bmal knockout mice have a range of phenotypes includingdecrease life span, low fertility, low body weight, age-dependentarthropathy, and brain astrogliosis.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is providedan inner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein and/or a circadian clock protein for use in theprevention and/or treatment of age-related macular degeneration.

Optionally, there is provided an inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein for use in theprevention and/or treatment of age-related macular degeneration.

Optionally or additionally, there is provided a circadian clock proteinfor use in the prevention and/or treatment of age-related maculardegeneration.

Optionally, there is provided an inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and a circadian clockprotein for use in the prevention and/or treatment of age-relatedmacular degeneration.

According to a second aspect of the present invention, there is provideda method for the prevention and/or treatment of age-related maculardegeneration in a subject, the method comprising the step ofadministering an inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or a circadianclock protein to the subject.

Optionally, the method comprises the step of administering aninner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein to the subject.

Optionally or additionally, the method comprises the step ofadministering a circadian clock protein to the subject.

Optionally, the method comprises the step of administering aninner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein and a circadian clock protein to the subject.

According to a third aspect of the present invention, there is provideduse of an inner-blood-retinal-barrier (iBRB) or blood-brain-barrier(BBB) tight junction protein and/or a circadian clock protein in themanufacture of a medicament for the prevention and/or treatment ofage-related macular degeneration.

Optionally, there is provided use of an inner-blood-retinal-barrier(iBRB) or blood-brain-barrier (BBB) tight junction protein in themanufacture of a medicament for the prevention and/or treatment ofage-related macular degeneration.

Optionally or additionally, there is provided use of a circadian clockprotein in the manufacture of a medicament for the prevention and/ortreatment of age-related macular degeneration.

Optionally, there is provided use of an inner-blood-retinal-barrier(iBRB) or blood-brain-barrier (BBB) tight junction protein and acircadian clock protein in the manufacture of a medicament for theprevention and/or treatment of age-related macular degeneration.

According to a fourth aspect of the present invention, there is providedan expression vector comprising a nucleic acid sequence encoding aninner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein and/or a nucleic acid sequence encoding a circadianclock protein for use in the prevention and/or treatment of age-relatedmacular degeneration.

According to a fifth aspect of the present invention, there is provideda composition comprising an inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or a circadianclock protein for use in the prevention and/or treatment of age-relatedmacular degeneration.

Optionally or additionally, the composition comprises an expressionvector comprising a nudeic acid sequence encoding aninner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein and/or a nucleic acid sequence encoding a circadianclock protein.

Optionally, the composition comprises an inner-blood-retinal-barrier(iBRB) or blood-brain-barrier (BBB) tight junction protein and/or acircadian clock protein; and an expression vector comprising a nucleicacid sequence encoding an inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or a nucleic acidsequence encoding a circadian clock protein.

Optionally, the use or administration restores the natural circadiancycling of the proteins in the iBRB.

Further optionally, the use or administration restores the naturalcircadian cycling of the proteins in the iBRB over a period of less than24 hours.

Alternatively, the use or administration restores the natural circadiancycling of the proteins in the iBRB over a period of 24 hours.

Further alternatively, the use or administration restores the naturalcircadian cycling of the proteins in the iBRB over a period of more than24 hours.

Optionally, the use or administration comprises increasing the amount ofthe inner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB)tight junction protein over a period of less than 24 hours, 24 hours, ormore than 24 hours.

Optionally or additionally, the use or administration comprisesdecreasing the amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein over a period of lessthan 24 hours, 24 hours, or more than 24 hours.

Optionally, the use or administration comprises sequentially increasingand decreasing the amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein over a period of lessthan 24 hours, 24 hours, or more than 24 hours.

Optionally or additionally, the use or administration comprisesincreasing the amount of the circadian clock protein over a period ofless than 24 hours, 24 hours, or more than 24 hours.

Optionally or additionally, the use or administration comprisesdecreasing the amount of the circadian clock protein over a period ofless than 24 hours, 24 hours, or more than 24 hours.

Optionally, the use or administration comprises sequentially increasingand decreasing the amount of the circadian clock protein over a periodof less than 24 hours, 24 hours, or more than 24 hours.

Optionally, the use or administration comprises increasing theexpression of a nucleic acid encoding the inner-blood-retinal-barrier(iBRB) or blood-brain-barrier (BBB) tight junction protein over a periodof less than 24 hours, 24 hours, or more than 24 hours.

Optionally or additionally, the use or administration comprisesdecreasing the expression of a nucleic acid encoding theinner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein over a period of less than 24 hours, 24 hours, or morethan 24 hours.

Optionally, the use or administration comprises sequentially increasingand decreasing the expression of a nucleic acid encoding theinner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein over a period of less than 24 hours, 24 hours, or morethan 24 hours.

Optionally or additionally, the use or administration comprisesincreasing the expression of a nucleic acid encoding the circadian clockprotein over a period of less than 24 hours, 24 hours, or more than 24hours.

Optionally or additionally, the use or administration comprisesdecreasing the expression of a nucleic acid encoding the circadian clockprotein over a period of less than 24 hours, 24 hours, or more than 24hours.

Optionally, the use or administration comprises sequentially increasingand decreasing the expression of a nucleic acid encoding the circadianclock protein over a period of less than 24 hours, 24 hours, or morethan 24 hours.

Optionally, the use or administration comprises increasing the amount ofthe inner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB)tight junction protein over a first period of less than 12 hours, 12hours, or more than 12 hours.

Optionally or additionally, the use or administration comprisesdecreasing the amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein over a second period ofless than 12 hours, 12 hours, or more than 12 hours.

Optionally, the use or administration comprises sequentially increasingthe amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein over a first period ofless than 12 hours, 12 hours, or more than 12 hours; and decreasing theamount of the inner-blood-retinal-barrier (iBRB) or blood-brain-barrier(BBB) tight junction protein over a second period of less than 12 hours,12 hours, or more than 12 hours.

Optionally or additionally, the use or administration comprisesincreasing the amount of the circadian clock protein over a first periodof less than 12 hours, 12 hours, or more than 12 hours.

Optionally or additionally, the use or administration comprisesdecreasing the amount of the circadian clock protein over a first periodof less than 12 hours, 12 hours, or more than 12 hours.

Optionally, the use or administration comprises sequentially increasingthe amount of the circadian clock protein over a first period of lessthan 12 hours, 12 hours, or more than 12 hours; and decreasing theamount of the circadian clock protein over a second period of less than12 hours, 12 hours, or more than 12 hours.

Optionally, the use or administration comprises increasing theexpression of a nucleic acid encoding the inner-blood-retinal-barrier(iBRB) or blood-brain-barrier (BBB) tight junction protein over a firstperiod of less than 12 hours, 12 hours, or more than 12 hours.

Optionally or additionally, the use or administration comprisesdecreasing the expression of a nucleic acid encoding theinner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein over a second period of less than 12 hours, 12 hours,or more than 12 hours.

Optionally, the use or administration comprises sequentially increasingthe expression of a nucleic acid encoding theinner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein over a first period of less than 12 hours, 12 hours, ormore than 12 hours; and decreasing the expression of a nucleic acidencoding the inner-blood-retinal-barrier (iBRB) or blood-brain-barrier(BBB) tight junction protein over a second period of less than 12 hours,12 hours, or more than 12 hours.

Optionally or additionally, the use or administration comprisesincreasing the expression of a nucleic acid encoding the circadian clockprotein over a first period of less than 12 hours, 12 hours, or morethan 12 hours.

Optionally or additionally, the use or administration comprisesdecreasing the expression of a nucleic acid encoding the circadian clockprotein over a second period of less than 12 hours, 12 hours, or morethan 12 hours.

Optionally, the use or administration comprises sequentially increasingthe expression of a nucleic acid encoding the circadian clock proteinover a first period of less than 12 hours, 12 hours, or more than 12hours; and decreasing the expression of a nucleic acid encoding thecircadian clock protein over a second period of less than 12 hours, 12hours, or more than 12 hours.

Optionally, the use or administration comprises increasing or decreasingthe amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein or the expression ofthe nucleic acid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein relative to a normalcontrol.

Optionally or additionally, the use or administration comprisesincreasing or decreasing the amount of the circadian clock protein orthe expression of the nucleic acid encoding the circadian clock proteinrelative to a normal control.

Optionally, the use or administration comprises increasing or decreasingthe amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and the circadian clockprotein; or increasing or decreasing the expression of the nucleic acidencoding the inner-blood-retinal-barrier (iBRB) or blood-brain-barrier(BBB) tight junction protein and the circadian clock protein; eachrelative to a normal control.

Optionally, a normal control is an amount of theinner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein and/or the circadian clock protein in a healthy subjector a subject not suffering from age-related macular degeneration,dry-AMD, wet-AMD, and/or geographic atrophy (GA).

Optionally, a normal control is an amount of the expression of thenucleic acid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the expressionof the nucleic acid encoding the circadian clock protein in a healthysubject or a subject not suffering from age-related maculardegeneration, dry-AMD, wet-AMD, and/or geographic atrophy (GA).

Optionally, a normal control is an amount of theinner-blood-retinal-barrier (iBRB) or blood-brain-barrier (BBB) tightjunction protein and/or the circadian clock protein in a precedingperiod of less than 12 hours, 12 hours, or more than 12 hours.

Optionally, a normal control is an amount of the expression of thenucleic acid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the expressionof the nucleic acid encoding the circadian clock protein in a precedingperiod of less than 12 hours, 12 hours, or more than 12 hours.

Optionally, the use or administration comprises increasing or decreasingthe amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; or increasing or decreasing the expression of the nucleicacid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; each by up to 50% relative to a normal control.

Further optionally, the use or administration comprises increasing ordecreasing the amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; or increasing or decreasing the expression of the nucleicacid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; each by up to 50%, optionally up to 40%, optionally up to30%, optionally up to 20%, optionally up to 10%, relative to a normalcontrol.

Optionally, the use or administration comprises increasing or decreasingthe amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; or increasing or decreasing the expression of the nucleicacid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; each by more than 50% relative to a normal control.

Further optionally, the use or administration comprises increasing ordecreasing the amount of the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; or increasing or decreasing the expression of the nucleicacid encoding the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein; each by more than 50%, optionally more than 60%,optionally more than 70%, optionally more than 80%, optionally more than90%, relative to a normal control.

Optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein is for use in the prevention and/or treatment of dry-AMD.

Optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein is for use in the prevention and/or treatment of wet-AMD.

Optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein and/or the circadianclock protein is for use in the prevention and/or treatment ofgeographic atrophy (GA).

Optionally, there is provided an inner blood retinal barrier(iBRB)/blood brain barrier (BBB) tight junction protein and/or acircadian clock protein that affects iBRB/BBB tight junction proteinexpression, for use in the prevention and/or treatment of age-relatedmacular degeneration, wherein the treatment involves restoring thenatural circadian cycling of the proteins in the iBRB over a 24 hourperiod. Preferably, the protein is for use in the treatment and/orprevention of dry-AMD, more preferably geographic atrophy.

Optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein is selected from theclaudin family of proteins.

Further optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein is selected from aclaudin-1, claudin-3, claudin-5, claudin-12 protein, and combinationseach thereof.

Still further optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein is a claudin-5 protein.

Optionally or additionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein is selected fromoccludin, tricellulin, Lipolysis-stimulated lipoprotein receptor (LSR),zonula occluden-1 (ZO-1), junctional adhesion molecule (JAM), andcombinations each thereof.

Optionally, the inner-blood-retinal-barrier (iBRB) orblood-brain-barrier (BBB) tight junction protein is selected fromclaudin-1, claudin-3, claudin-5, claudin-12, occludin, tricellulin,Lipolysis-stimulated lipoprotein receptor (LSR), zonula occluden-1(ZO-1), junctional adhesion molecule (JAM), and combinations eachthereof.

Optionally, the circadian clock protein is selected from period-1(Per-1), period-2 (Per-2), period-3 (Per-3), cryptochrome-1 (Cry-1),cryptochrome-2 (Cry-2), Clock, brain and muscle aryl hydrocarbonreceptor nuclear translocator like-1 (BMAL-1), brain and muscle arylhydrocarbon receptor nuclear translocator like-2 (BMAL-2), Rev-ErbAalpha (NR1D1), and combinations each thereof.

Optionally, the circadian clock protein is selected from period-2(Per-2), brain and muscle aryl hydrocarbon receptor nuclear translocatorlike-1 (BMAL-1), Rev-ErbA alpha (NR1D1), and combinations each thereof.

Optionally, there is provided a claudin-5 protein and/or a circadianclock protein that affects claudin-5 expression for use in theprevention and/or treatment of geographic atrophy (GA) associated withdry age-related macular degeneration (dry AMD), wherein the treatmentinvolves restoring the natural circadian cycling of the proteins in theiBRB over a 24 hour period.

Optionally, there is provided an expression vector comprising aclaudin-5 nucleic acid sequence or fragment thereof and/or a circadianclock nucleic acid sequence or fragment thereof that affects claudin-5expression for use in the treatment of geographic atrophy (GA)associated with dry age-related macular degeneration (dry AMD), whereinthe treatment involves restoring the natural circadian cycling of theproteins in the iBRB over a 24 hour period.

Optionally, there is provided a pharmaceutical composition comprising aclaudin-5 protein or expression vector comprising a claudin-5 nucleicacid sequence or fragment thereof and/or a circadian clock protein thataffects claudin-5 expression or expression vector a circadian clocknucleic acid sequence or fragment thereof that affects claudin-5expression, for use in the prevention and/or treatment of associatedwith dry age-related macular degeneration (dry AMD), preferablygeographic atrophy (GA), wherein the treatment involves restoring thenatural circadian cycling of the proteins in the iBRB over a 24 hourperiod.

Optionally, there is provided a method for the prevention and/ortreatment of age-related macular degeneration, wherein the treatmentinvolves restoring the natural circadian cycling of the inner bloodretinal barrier (iBRB) tight junction proteins, preferably claudin-5,and/or the circadian clock proteins that affects iBRB tight junctionprotein expression, preferably claudin-5, over a 24 hour period.

DETAILED DESCRIPTION OF THE INVENTION

In this specification, we have described the treatment in terms ofrestoring the natural circadian cycling of the proteins of interest inthe iBRB and/or the BBB over a 24 hour period. The term ‘naturalcycling’ or ‘natural circadian cycling’ is intended to beinterchangeable with the terms ‘dynamic cycling’ or ‘dynamic circadiancycling’, ‘circadian rhythm expression’ and the like.

According to a general aspect of the invention, there is provided aninner blood retinal barrier (iBRB) tight junction protein and/or acircadian clock protein that affects iBRB tight junction proteinexpression, for use in the prevention and/or treatment of age-relatedmacular degeneration, wherein the treatment involves restoring thenatural circadian cycling of the proteins in the iBRB over a 24 hourperiod.

Preferably, the protein is for use in the treatment and/or prevention ofdry AMD, more preferably geographic atrophy.

This general aspect of the invention is based on the findings thatclaudin-5, one of the inner blood retinal barrier (iBRB) tight junctionproteins, surprisingly cycles at the BBB/iBRB. We have surprisinglydemonstrated that the expression of claudin-5 is affected by circadianclock proteins that affect iBRB tight junction protein expression. Thesefinding are entirely unexpected, as to date the iBRB and BBB areaccepted as “static” environments. We have also shown there is aphenotypic correlation to this cycling (see FIGS. 10 and 11) and thatpersistent suppression of claudin-5 induces a GA phenotype.

Based on these findings, the invention is directed to there-establishment of inner blood retinal barrier (iBRB) tight junctionprotein “cycling” and/or a circadian clock protein that affects iBRBtight junction protein “cycling” to treat AMD, particular dry-AMD, endstage dry AMD/GA.

The treatment involves restoring the circadian rhythm and/orresynchronizing the biological clock of the iBRB. The inventors havesurprisingly found that re-establishing or restoring the natural cyclingof the proteins of interest, particularly claudin-5, at the iBRB willregulate the passive diffusion of material into the photoreceptor outersegments and prevent the development of AMD, particularly late-stageAMD, geographic atrophy (GA).

Thus, the general aim is to maintain the inner blood retinal barrier(iBRB) tight junction protein and/or a circadian clock proteinexpression levels at normal levels over a 24 hour period and due to thecycling of these proteins, the method essentially aims to mimic,reinstate or restore the natural cycling/circadian rhythm expression ofthese proteins. Thus, the treatment covers the restoration orre-establishment of the natural or dynamic cycling of the proteins ofinterest where evening/night-time levels (from approximately 8 pm to 8am) of the proteins of interest, particularly claudin-5 are decreasedcompared to morning/daytime levels.

In this manner, the levels of the proteins of interest, particularlyclaudin-5, can be upregulated and/or downregulated over a 24 hour periodin order to restore the natural circadian rhythm of the iBRB andexpression of the proteins of interest.

According to an aspect of the invention, there is provided a claudin-5protein and/or a circadian clock protein that affects claudin-5expression for use in the prevention and/or treatment of geographicatrophy (GA) associated with dry age-related macular degeneration (dryAMD), wherein the treatment involves restoring the natural circadiancycling of the proteins in the iBRB over a 24 hour period.

The inner blood-retinal barrier (inner BRB/iBRB) is created by complextight junctions of retinal capillary endothelial cells. Although thisbarrier prevents the free diffusion of substances between thecirculating blood and the neural retina, the inner BRB efficientlysupplies nutrients to the retina.

In this manner, the iBRB tight junction protein, such as claudin-5, is acomponent of the inner retinal vasculature and is proposed as atherapeutic target for GA treatment. This is in contrast to the knownsuggested approaches to treating GA which involve targeting the RPEdirectly. The present invention advantageously provides a method for theearly stage intervention treatment of dry AMD.

It is postulated that regulating levels of claudin-5 at the iBRB conferscontrol over the delivery and replenishment of the outer segments ofphotoreceptors and consequently control the burden of material consumedby the RPE on a daily basis. Accordingly, Claudin-5 is proposed as apreferred target as it is the main component of the iBRB tight junctioncomplex. Other tight junction proteins present in the iBRB tightjunction complex include Occludin, claudin-1, -3, -12, tricellulin, LSR,ZO-1, junctional adhesion molecule (JAM) and could be additional (singleor combination) targets for the proposed therapy.

The mammalian circadian clock in the neurons of suprachiasmatic nuclei(SCN) in the brain and in cells of peripheral tissues is driven by aself-sustained molecular oscillator, which generates rhythmic geneexpression with a periodicity of about 24. 24 hour circadian cyclecontrolled by the oscillatory expression of a clock gene cassetteincluding Period (Per) 1-3, Cryptochrome (Cry) 1/2, Clock, and Bmal1/2.The preferred circadian clock proteins that affects iBRB tight junctionprotein expression include Period (Per) 1-3, Cryptochrome (Cry) 1/2,Clock, and Bmal1/2,preferably BMAl1, Rev-Erb-alpha and/or Per2.

According to a preferred embodiment of the invention, there is provideda claudin-5 protein and/or a circadian clock protein that affectsclaudin-5 expression for use in the prevention and/or treatment ofgeographic atrophy (GA) associated with dry age-related maculardegeneration (dry AMD), wherein the treatment involves restoring thenatural circadian cycling of the claudin-5 protein in the iBRB over a 24hour period.

It will be understood that the subject has a geographic atrophyphenotype or is a subject at risk of developing geographic atrophy,which can be based on current diagnostic methods, including determinedarea and size of drusen deposition that will lead to a diagnosis of dryAMD in the first instance.

In general terms, the treatment involves any means by which the naturalcircadian cycling of the proteins in the iBRB over a 24 hour period isrestored and, for example, may take place by constitutivelyover-expressing the protein (or a vector comprising nucleic acidsequences) combined with the periodic suppression over a 24 hour period;or regulating the protein expression (or a vector comprising nucleicacid sequences) in a phased manner over a 24 hour period.

According to an aspect of the invention, there is provided an expressionvector comprising claudin-5 nucleic acid sequences and/or a circadianclock nucleic acid sequences that affects claudin-5 expression for usein the treatment of geographic atrophy (GA) associated with dryage-related macular degeneration (dry AMD), wherein the treatmentinvolves restoring the natural circadian cycling of the proteins in theiBRB over a 24 hour period.

Ideally, the nucleic acid is cDNA.

According to an aspect of the invention, there is provided apharmaceutical composition comprising a claudin-5 protein or expressionvector comprising claudin-5 nucleic acid sequences and/or a circadianclock protein that affects claudin-5 expression or expression vectorcomprising a circadian clock nucleic acid sequence that affectsclaudin-5 expression, for use in the prevention and/or treatment ofgeographic atrophy (GA) associated with dry age-related maculardegeneration (dry AMD), wherein the treatment involves restoring thenatural circadian cycling of the proteins in the iBRB over a 24 hourperiod.

According to all aspects and embodiments of the invention, the protein,expression vector, pharmaceutical composition may be provided in a formsuitable for systemic, intravitreal or sub-retinal delivery.

In this manner, the treatment may be in the form of small molecules,recombinant proteins, antibodies, siRNAs or viral vectors.

It will be understood that the cycling of the inner blood retinalbarrier (iBRB) tight junction protein, such as claudin-5, may beregulated directly.

Alternatively, the cycling of the inner blood retinal barrier (iBRB)tight junction protein, such as claudin-5, may be regulated indirectlyby targeting circadian clock protein that affects iBRB tight junctionprotein expression. By targeting the circadian dock proteins, thedownstream tight junction proteins can also be regulated.

Non-limiting and exemplary ways of direct and indirect targetinginclude:

-   -   a cDNA and/or shRNA based construct that will allow for        constitutive over-expression of any one of claudin-5, BMAL-1,        Reverb-alpha or Per2 while periodically allowing for its        suppression using, for example, doxycycline inducible promoters        driving claudin-5 shRNA or BMAL-1, or Reverb-alpha or Per2        shRNAs;    -   RNA interference (RNAi) agents, particularly siRNAs, can be        delivered to the target cell exogenously or expressed        endogenously in the form of short hairpin RNAs (shRNAs). In        shRNA, the single RNA strand may form a hairpin structure with a        stem and loop and, optionally, one or more unpaired portions at        the 5′ and/or 3′ portion of the RNA;    -   periodic suppression is required as the method involves the        constitutive over-expression of claudin-5 via cDNA based        constructs. Periodic suppression of claudin-5 will restore the        “cycling” of the protein again;    -   a regulatable claudin-5, BMAL-1, Reverb-alpha or Per2 cDNA        expression vector that provides “phased” expression of the        proteins;    -   ideally, the construct is doxycline regulatable. Other promoters        and drugs may be used also. Ideally, this construct provides for        phased daily exposure to doxycycline at the same time each day.    -   a pulsatile exposure to dexamethsone which can directly regulate        claudin-5 or BMAL-1, or Reverb-alpha or Per2 levels in the eye;    -   ideally, this involves a slow release encapsulation device        implanted into the eye or subcutaneously that will allow for a        distinct amount of dexamethasone to be released daily at the        same time. In this manner dexamethasone, which is know to        regulate tight junctions, can be used in a pulsatile manner to        phenocopy circadian cycling of claudin-5.

According an aspect of the invention, there is provided a method for theprevention and/or treatment of age-related macular degeneration, whereinthe treatment involves restoring the natural circadian cycling of theinner blood retinal barrier (iBRB) tight junction proteins and/or thecircadian clock 5 protein that affects iBRB tight junction proteinexpression over a 24 hour period.

Ideally, the method is for the prevention and/or treatment of geographicatrophy (GA) associated with dry age-related macular degeneration (dryAMD).

According to a preferred embodiment, the method involves the regulationof inner blood retinal barrier (iBRB) tight junction protein, such asclaudin-5, and/or the regulation of circadian clock protein that affectsiBRB tight junction protein expression.

As defined above, the iBRB tight junction proteins can be selected fromone or more of occludin, claudin-1, claudin-3, claudin-5, claudin-12,tricellulin, LSR, ZO-1 and/or junctional adhesion molecule (JAM).

As defined above, the circadian clock protein that affects iBRB tightjunction protein expression can be selected from one or more of Period(Per) 1-3, Cryptochrome (Cry) 1/2, Clock, and Bmal1/2, preferably BMAI1,Rev-Erb-alpha and/or Per2.

According to a preferred embodiment, the protein is claudin-5 and/or acircadian clock protein that affects claudin-5 expression.

According to a more preferred embodiment, the method is the preventionand/or treatment of geographic atrophy (GA) associated with dryage-related macular degeneration (dry AMD). In this manner, the subjecthas a geographic atrophy phenotype or is at risk of developing GA basedon determined area and size of drusen deposition.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thefollowing non-limiting examples and drawings, in which:

FIG. 1 illustrates retina, heart, and liver RNA isolated from 3-monthold C57BL/6J mice at 8 and 8 pm, and transcript levels of claudin-5analysed by SYBR green RT-PCR, wherein β-actin was used as ahousekeeping gene reference for standardisation (*P<0.05);

FIG. 2 illustrates a) retinae isolated from 3-month old C57BL/6J mice at8 am and 8 pm that had been housed in normal light-dark conditions,wherein protein lysates were analysed by western blotting to determinethe levels of claudin-5, with β-actin used as a loading control; b)densitometric quantification of claudin-5 expression showing asignificant decrease at 8 pm relative to 8 am; c) exposure of mousebrain endothelial cells to 50% serum for 2 hours followed by serum-freemedia for the length of times indicated inducing claudin-5 cycling,wherein data are representative from 5 individual mice retinae andanalysed by Student's t-test (* P value<0.05);

FIG. 3 illustrates a) retinae isolated from 3-month old C57BL/6J mice at8 am and 8 pm that had been housed in 24 h darkness (DA) to see if theresponse was circadian or diurnal (i.e. due to light), and transcriptlevels of claudin-5 analysed by SYBR green RT-PCR, wherein β-actin wasused as a house-keeping gene for standardisation; b) retinae isolatedfrom 3-month old C57BL/6J mice that had their circadian rhythm inversedfor 3 weeks (i.e. for these mice 8 am becomes 8 pm, 8 pm becomes 8 am)and compared to mice that had been kept on their normal light cycle, andclaudin-5 transcript levels analysed as mentioned for a), wherein dataare representative of retinae from 5 individual mice and analysed byStudent's t-test (* P value<0.05);

FIG. 4 illustrates a) retina RNA isolated from 3-month old C57BL/6J miceat 8 am and 8 pm and transcript expression for the dock componentsBmal1, Period 2 and Rev-Erbα analysed by SYBR green RT-PCR; b)transcript levels of Bmal1, Period 2, and Rev-Erba analysed from retinalRNA of mice that had been kept in 24 hours of darkness, wherein data arerepresentative of retinae from 5 individual mice and analysed byStudent's t-test (* P value<0.05, ** P value<0.01);

FIG. 5 illustrates fundus fluorescein angiography carried out at 8 amand 8 pm on C57BL/6J mice, wherein equal volume/weight of sodiumfluorescein was injected per mouse, and two minutes post-injectionimages were taken every 30 seconds up to 10 minutes with the sensitivityof the images being kept the same at each time point, wherein therelative image density was analysed using Image J software andrepresentative images at both 8 am and 8 pm are shown at 3, 5 and 10 minpost injection, and wherein the top right hand graph shows the averageimage raw intensity density for the entire image across all images andtime points, and the bottom right hand graph shows the area under thecurve;

FIG. 6 illustrates fundus fluorescein angiography carried out at 8 amand 8 pm on a) 129 mice and b) CD1 mice in the same manner as describedin FIG. 3, wherein representative images at both 8 am and 8 pm are shownat 3, 5 and 10 min post injection, and the graph to the right shows theaverage raw intensity density for microvessel permeability across allimages and time points, wherein data are representative of at least 5mice imaged at both time points and analysed by two-way ANOVA (* Pvalue<0.05, ** P value<0.01, *** P value<0.001);

FIG. 7 illustrates BMAL1^(FL/FL)×Tie2Cre⁺ imaged at 8 am and 8 pm byfundus fluorescein angiography as previously described, whereinrepresentative images from the same mouse imaged at both time points at3, 5 and 10 min post-injection are shown, and the graph below showsmicrovessel permeability of all images across all time points, and thereis no change in permeability between 8 am and 8 pm in mice lacking BMAL1in their endothelial cells, wherein data are representative of 12 micethat were all imaged at both time points and analysed by two-way ANOVA(ns, P value>0.05);

FIG. 8 illustrates BMAL1^(WT/FL)×Tie2Cre⁺ imaged at 8 am and 8 pm byfundus fluorescein angiography as previously described, whereinrepresentative images from the same mouse imaged at both time points at3, 5 and 10 min post-injection are shown, and the graph below shows thatthere is no change in permeability between 8 am and 8 pm in mice lackingjust one copy of BMAL1 in their endothelial cells, wherein data arerepresentative of 12 mice that were all imaged at both time points andanalysed by two-way ANOVA (ns, P value>0.05);

FIG. 9 illustrates inner blood-retinal barrier integrity in the morningand evening as studied by magnetic resonance imaging (MRI), wherein3-month old C57BU6J mice were injected via tail-vein with gadoliniumdiethylene-triamine penta-acetic acid (Gd-DTPA), and signal intensityfor both the left and right eye were analysed, wherein data arerepresentative of 5 mice and analysed by two-way ANOVA with Bonferronipost-hoc test (*** P value<0.001);

FIG. 10 illustrates NT AAV injected eye on normal diet (ND), top leftpanel; CL5 MV injected eye on ND, top right panel; NT AAV injected eyeon high fat diet (HFD), bottom left panel; CL5 AAV injected eye on HFD,bottom right panel;

FIG. 11 illustrates retinal cryosection of NT MV injected eye of mouseon high fat diet (HFD), top left panel; retinal cryosection of CL5injected eye of mouse on HFD, top right panel; retinal cryosection ofdonor human eye, bottom left panel; retinal cryosection of donor humaneye with geographic atrophy (GA);

FIG. 12 illustrates inducible Claudin-5×Tie2Cre mice that had theirwater supplemented with doxycycline to induce claudin-5 suppression fortwo weeks, and fundus fluorescein angiography carried out on mice thatexpress Tie2Cre (Tie2Cre+) leading to suppressed claudin-5 levels or onTie2Cre negative (Tie2Cre−) littermate controls in which claudin-5levels remain unchanged, wherein equal volume/weight of sodiumfluorescein was injected per mouse, and two minutes post-injectionimages were taken every 30 seconds up to 10 minutes with the sensitivityof the images being kept the same at each time point, and the relativeimage density analysed using Image J software, whereinCldn5-160×Tie2Cre+ have a more permeable retinal vasculature compared tolittermate controls, and wherein representative images from one mouse ofeach genotype are shown at 3, 5 and 10 min post injection, and the graphbelow shows the average image raw intensity density for themicrovasculature across all images and time points, wherein data arerepresentative of 5 mice imaged for both genotypes and analysed bytwo-way ANOVA (p value *** <0.0001);

FIG. 13 illustrates C57BU6J mice injected intravitreally with either anon-targeting (NT) or claudin-5 (Cldn5) siRNA, and fundus fluoresceinangiography (FFA) carried out 24, 48, 72 and 96 hour post-injection tostudy inner blood-retinal barrier permeability as previously described,wherein a representative image from an individual mouse is shown fromeach time point and for each siRNA injected (3-4 mice were injected persiRNA group per time point);

FIG. 14 illustrates C57BL/6J mice sacrificed at either 8 AM or 8 PM,wherein eyes were collected, fixed, enucleated and processed forelectron microscopy analysis to look at retinal endothelial cellintegrity, wherein boxes indicate regions of increased magnification,and tight junctions (as indicated by the arrow) appear to be morediffuse and less structured at 8 PM compared to 8 AM; and

FIG. 15 illustrates Bmal1FL/WT×Tie2Cre mice placed on a high fat diet(HFD) for 1 week and then imaged by fundus fluorescein angiography tostudy retinal vasculature permeability as previously described, whereinmice that are Tie2Cre+ lack one copy of Bmal1 in their retinalendothelial cells, and Bmal1FL/WT×Tie2Cre+ mice on HFD have a morepermeable retinal vasculature when compared to their littermate Tie2Cre−controls, wherein a representative image is shown from 3, 5 and 10 minpost-injection for both Bmal1FL/WT×Tie2Cre+ and Bmal1FL/WT×Tie2Cre− miceon HFD, and the graph below shows the relative image density for themicrovasculature for both genotypes across all time points, which wasanalysed using Image J software, and wherein data are representative of6-8 mice imaged for both genotypes and analysed by two-way ANOVA (pvalue * 0.04).

EXAMPLES Material & Methods

AAV production

shRNAs designed to target transcripts derived from mouse claudin-5 wereincorporated into AAV-2/9 vectors. shRNA was cloned into thepSingle-tTS-shRNA (Clontech) vector in accordance with themanufacturer's instructions. The plasmid incorporating the induciblesystem with claudin-5 shRNA was digested with BsrBi and BsrGl andligated into the Not1 site of the plasmid pAAV-MCS, such as toincorporate left and right AAV inverted terminal repeats (L-ITR andR-ITR). AAV-2/9 was then generated using a triple transfection system ina stably transfected HEK-293 cell line for the generation of high-titreviruses (Vector BioLabs).

Magnetic Resonance Imaging (MRI)

BRB integrity was assessed in vivo via MRI, using a dedicated smallrodent 7 T MRI system located at TCD(www.neuroscience.tcd.ie/technologies/mri.php). Anaesthetised mice werephysiologically monitored (ECG, respiration and temperature) and placedon an MRI-compatible support cradle, with a built-in system formaintaining the animal's body temperature at 37° C. The cradle was thenpositioned within the MRI scanner. Accurate positioning was ensured byacquiring an initial rapid pilot image, which was then used to ensurethe correct geometry was scanned in all subsequent MRI experiments. Uponinsertion into the MRI scanner, high resolution anatomical images of thebrain were acquired (100 μm in-plane and 500 μm through-plane spatialresolution). To visualize brain damage and lesion volumes, highresolution images were acquired using Rapid Acquisition with RelaxationEnhancement (RARE) 2-D sequence with a RARE factor of 8 and an echo timeresulting in an effective time of 42.2 ms (with a flip angle of 180°).With an acquisition matrix of 128×128 and a field of view of 1.8×1.8cm², the pixel resolution was 0.141 mm/pixel. In the coronal plane, 15slices, each measuring 0.25 mm in thickness were acquired. Repetitiontime was 7274.2 ms, and four averages were used for a total measuringtime of 7 minutes 45 seconds.

Compromises of the BRB were then visualised in high resolution T1weighted MR images (resolution, 0.156×0.156×5 mm³; field of view:20×20×17.9 mm³; matrix; 128×128×30; TR/TE: 500/2.7 ms; flip angle: 30°;number of averages: 3; acquisition time: 2 min, 24 sec; Repetitions: 12)following administration of 100 μl of a 1 in 3 dilution of Gd-DTPA(Gadolinium diethylene-triamine penta-acetic acid), administered via thetail vein.

Real-Time RT-PCR Analysis

Transcript levels were quantified using a two-step real-time reversetranscription polymerase chain reaction (RT-PCR) on the 7300 Real-TimePCR System (Applied Biosystems) with QuantiTect SYBR Green I (Qiagen) asa fluorescent dye. cDNA was reverse transcribed from RNA with theHigh-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Real-time PCR was performed with the FastStart Universal SYBR GreenMaster (ROX) master mix (Roche). The RT-PCR reaction conditions were asfollows: 95° C.×10 min, (95° C.×10 s, 60° C.×30 s)×40, 95° C.×15 s, 60°C.×1 min, 95° C.×15 s, 60° C.×15 s. The primer sequences for the RT-PCRexperiments were supplied by Sigma-Aldrich and were as follows:claudin-5 left, 5′-TTTCTTCTATGCGCAGTTGG-3′, and right,5′-GCAGTTTGGTGCCTACTTCA-3′: β-actin left,5′-TCACCCACACTGTGCCCATCTACGA-3′ and right,5′-CAGCGGAACCGCTCATTGCCAATGG-3′. Relative gene expression levels weremeasured using the comparative C_(T) method (ΔΔC_(T)). Expression levelsof target genes were normalised to the housekeeping gene β-actin.

Cell Culture and Transfection

Mouse brain endothelial cells (Bend.3, American Type Culture Collection)were cultured in DMEM supplemented with 10% FCS and 2 mM sodium pyruvatein a 5% CO₂ incubator at 37° C. Bend.3 cells were seeded on 12 wellplates (2.5*10⁵ cells per well) and 100 ng/ml claudin-5 shRNA wastransfected per well using Lipofectamine 2000. RNA was extracted fromHEK293 cells and Bend.3 cells with the E.Z.N.A Total RNA Kit 1 (Omegabiotek) according to the manufacturer's instructions. Proteins wereisolated with lysis buffer (62.5 mM Tris, 2% SDS, 10 mM Dithiothreitol,10 μl protease inhibitor cocktail/100 ml (Sigma Aldrich), followed bycentrifugation at 12,000 RPM for 20 min @ 4° C. and supernatant wasremoved for claudin-5 protein analysis.

Fundus Fluorescein Angiography (FFA)

Mice were prepared for FFA analysis by instillation oftropicamide/phenylephrine eye drops and subsequently anaesthetised usingKetamine/Domitor mix. The fundus was imaged using a Heidelberg opticalcoherence tomography (OCT) machine and retinal vasculature was assessedfollowing intra-peritoneal injection of a solution containing sodiumfluorescein (2%). Images were acquired every 30 seconds for a period of10 minutes.

Example 1—Claudln-5 is Regulated by the Circadian Clock

Circadian rhythms are 24 hour oscillations in behaviour and physiologyin response to environmental cues, primarily daylight and darkness. Aconserved transcriptional-translational regulatory loop involving coreclock components, gene protein products, are necessary for generationand regulation of circadian rhythms within individual cells. It wasdetermined that claudin-5 was cycling in a circadian rhythm in allorgans analysed (see FIG. 1). In tandem, claudin-5 protein levels aresignificantly decreased in the evening (8 PM) when compared to themorning (8 AM) in mice (see FIG. 2a, b ). While in vitro, cells losetheir circadian rhythm, following serum shock, it is possible to resetthe circadian clock transiently. In primary mouse brain endothelialcells, serum shock induced cycling of claudin-5 in 12 hour phasessimilar to that observed in the retina and peripheral organs (see FIG.2c ). This response is circadian rather than diurnally regulated asclaudin-5 levels remain lower in the evening compared to morning formice that had been kept in 24 hours of darkness (see FIG. 3a ).Circadian rhythms can be intrinsically inversed following 3 weeks oflight alteration and we found that mice that have an inversed circadianrhythm, meaning 8 am is actually 8 pm, have lower claudin-5 levels (seeFIG. 3b ). Clock components BMAL1, Per2 and Rev-Erb-alpha were found tonot cycle in an exact 24 hour cycle as expected (see FIG. 4a ) althoughBMAL1 and Rev-Erb-alpha do have lower expression levels at 8 pm in micethat have been kept in darkness for 24 hours (see FIG. 4b ).

Inner retinal blood vessels are more permeable in the evening comparedto the morning. Given the changes in expression of the key tightjunction component claudin-5 at various times of the day, retinal fundusfluroescein angiography (FFA) was performed at 8 AM compared to 8 PM.Retinal blood vessels appeared to be more “leaky” in the eveningcompared to the morning that correlated with the levels of claudin-5protein expression at these time points (see FIG. 5). This phenomenon isnot strain-specific as we also see ‘leakier’ vessels in the evening inboth 129 mice and the CD1 strain (see FIG. 6a, b ). Endothelial cellspecific suppression of BMAL1, using BMAL1FL/FL mice crossed to Tie2Cremice results in no changes in permeability at 8 am and 8 pm beingobserved (see FIG. 7). The loss of just one copy of BMAL1 appears to besufficient in preventing the permeability changes seen (see FIG. 8).This is suggestive of BMAL1 playing a role in regulating retinalpermeability changes. In corroboration, magnetic resonance imaging showsthat the retina is more permissive in the evening (see FIG. 9).

Example 2—Claudin-5 Suppression and Supplementation of Mice With a HighFat Diet Induces Geographic Atrophy-Like Phenotype

Given the phenotype observed with regard to enhanced fluroescein leakageat 8 PM, it was sought to induce this phenotype for a prolonged periodof time using an adeno-associated virus (MV) vector expressing claudin-5shRNA under the control of a doxycycline inducible promoter. To thisend, 10 C57BL/6 mice were injected sub-retinally with AAV-luciferaseinto their left eye and AAV2/9 claudin 5 into the right eye. Five micewere left on normal diet (ND) and the remaining five placed onto a highfat diet (HFD) with all mice on doxycycline water (2 mg/ml) to induceshRNA expression. Six weeks post-injection, FFA analysis showed enhancedleakage of fluorescien in the AAV2/9 claudin-5 eye compared to theAAV-luciferase injected eye (see FIG. 10). Post mortem analysis of eyesfrom these mice revealed a retinal pigment epithelium (RPE) phenotypesimilar to that observed in human subjects with GA (see FIG. 11).

Example 3—Inducible Claudin-5×Tie2Cre+ Mice Have a ‘Leakier’ InnerBlood-Retinal Barrier

As mice that are deficient in claudin-5 are embryonic lethal, a newmouse model was generated that allows for inducible suppression ofclaudin-5. These mice are then crossed to the Cre-recombinase expressing(Tie2Cre) mice and animals that are Cre positive have suppressed levelsof claudin-5 in their retinal endothelial cells when administereddoxycycline in drinking water. Claudin-5×Tie2Cre mice were administereddoxycycline for two weeks and then imaged by fundus fluoresceinangiography (FFA) (see FIG. 12). Inducible claudin-5×Tie2Cre+ mice havemore permeable retinal vessels when compared to their Tie2Cre−littermate controls.

Example 4—Administration of Claudin-5 siRNA Intravitreally Leads to MorePermeable Inner Retinal Blood Vessels

Given suppression of claudin-5 leads to more ‘leaky’ vessels, it wassought to determine the time frame of when maximum suppression ofclaudin-5 is observed. To this end, cohorts of 3-4 mice were injectedintravitreally with either a non-targeting (NT) or claudin-5 (Cldn5)siRNA and subsequently imaged 24, 48, 72 or 96 hours post-injection byFFA (see FIG. 13) prior to sacrifice for protein and transcriptanalysis. It appears within 24 hours mice injected with claudin-5 siRNAhave more permeable retinal vessels when compared to those injected withNT siRNA.

Example 5—Tight Junction Structure Appears to Differ at 8AM and 8PM inC57BL/6J Mice

Previous data have shown that the inner retinal vasculature is morepermeable in the evening compared to the morning by both FFA andmagnetic resonance imaging (MRI). To further investigate the tightjunction structure, C57BL/6J mice were sacrificed at 8AM and 8PM andeyes processed for electron microscopy analysis. The tight junctions at8AM appear more electron dense and defined when compared to 8PM whichare more diffuse and less structured in appearance (see FIG. 14).

Example 6—Supplementation of Bmal1×Tie2Cre Mice with a High Fat DietInduces ‘Leakier’ Inner Blood-Retinal Vessels

Given the phenotype observed with no change in enhanced fluoresceinleakage at 8 PM with Bmal1×Tie2Cre+ mice as seen in C57BL/6J mice, itwas sought to see if supplementation of a high fat diet (HFD) couldinduce enhanced fluorescein leakage. After one week of the mice being onHFD, FFA was carried out to study retinal vessel integrity.Bmal1×Tie2Cre+ mice lack one copy of the clock gene Bmal1 in theirendothelial cells. Bmal1×Tie2Cre+ mice on HFD have more permeableretinal vessels when compared to their littermate Bmal1×Tie2Cre−controls (see FIG. 15).

Together, these findings suggest claudin-5 and components of thecircadian clock are direct targets for therapeutic intervention in GA,as persistent suppression of claudin-5 induces a GA phenotype, thereforeregulating its levels can prevent the phenotype.

1-19. (canceled)
 20. A method for the treatment of age-related maculardegeneration in a subject, the method comprising the step of increasingor decreasing in the subject the amount of aninner-blood-retinal-barrier (iBRB) protein selected from claudin-1,claudin-3, claudin-12, occludin, tricellulin, Lipolysis-stimulatedlipoprotein receptor (LSR), zonula occluden-1 (ZO-1), junctionaladhesion molecule (JAM), and combinations each thereof; and a circadianclock proteinselected from period-1 (Per-1), period-2 (Per-2), period-3(Per-3), cryptochrome-1 (Cry-1), cryptochrome-2 (Cry-2), Clock, brainand muscle aryl hydrocarbon receptor nuclear translocator like-1(BMAL-1), brain and muscle aryl hydrocarbon receptor nucleartranslocator like-2 (BMAL-2), Rev-ErbA alpha (NR1 D1), and combinationseach thereof.
 21. The method according to claim 1, wherein the methodcomprises increasing the amount of the inner-blood-retinal-barrier(iBRB) protein over a period of 24 hours.
 22. The method according toclaim 1, wherein the method comprises decreasing the amount of theinner-blood-retinal-barrier (iBRB) protein over a period of 24 hours.23. The method according to claim 1, wherein the method comprisessequentially increasing and decreasing the amount of theinner-blood-retinal-barrier (iBRB) protein over a period of 24 hours.24. The method according to claim 1, wherein the method comprisesincreasing the amount of the circadian clock protein over a period of 24hours.
 25. The method according to claim 1, wherein the method comprisesdecreasing the amount of the circadian clock protein over a period of 24hours.
 26. The method according to claim 1, wherein the method comprisessequentially increasing and decreasing the amount of the circadian clockprotein over a period of 24 hours.
 27. The method according to claim 1,wherein the method comprises sequentially increasing the amount of theinner-blood-retinal-barrier (iBRB) protein over a first period of 12hours; and decreasing the amount of the inner-blood-retinal-barrier(iBRB) protein over a second period of 12 hours.
 28. The methodaccording to claim 1, wherein the method comprises sequentiallyincreasing the amount of the circadian clock protein over a first periodof 12 hours; and decreasing the amount of the circadian clock proteinover a second period of 12 hours.
 29. The method according to claim 1,wherein the method comprises increasing or decreasing the amount of theinner-blood-retinal-barrier (iBRB) protein and the circadian clockprotein, each relative to a normal control, wherein a normal control isan amount of the inner-blood-retinal-barrier (iBRB) protein and thecircadian clock protein in a healthy subject.
 30. The method accordingto claim 10, wherein the method comprises increasing or decreasing theamount of the inner-blood-retinal-barrier (iBRB) protein and thecircadian clock protein; each by up to 50% relative to the normalcontrol.
 31. The method according to claim 10, wherein the methodcomprises increasing or decreasing the amount of theinner-blood-retinal-barrier (iBRB) protein and the circadian clockprotein; each by more than 50% relative to the normal control.
 32. Themethod according to claim 1, wherein the age-related maculardegeneration is dry-AMD.
 33. The method according to claim 1, whereinthe age-related macular degeneration is geographic atrophy (GA).
 34. Themethod according to claim 1, wherein the inner-blood- retinal-barrier(iBRB) protein is selected from a claudin-1, claudin-3, claudin-12protein, and combinations each thereof.
 35. The method according toclaim 1, wherein the circadian clock protein is brain and muscle arylhydrocarbon receptor nuclear translocator like-1 (BMAL-1).